Extensive amounts of research and money are being invested to develop methods to sequence individual strands of DNA. However, in most proposed single molecule sequencing approaches, the overall rate of sequencing is limited by the rate of arrival of individual target molecules at the sequencing element. One way to increase the overall rate of arrival is to amplify the number of DNA molecules in the solution using a polymerase chain reaction or equivalent method. Such amplification is required for all existing DNA sequencing approaches and adds considerable cost and complexity, particularly if an entire human genome is to be amplified. To realize the true potential of such single molecule sequencing methods, the overall measurement approach should ideally not requite amplification of the original DNA sample.
For example, a good commercial DNA processing system can extract DNA equivalent to 30 million genomes from 100 μl of blood. A compact nanopore-base measurement system as recently described by Barrall et al., (Barrall et al. 2008, Ervin and Barrall 2007) has an analyte volume of order 50 micro liters (μl), resulting in an average of only 15 million genomes in the sensing volume that contains the nanopore. Scaling from capture rate data of short (˜1 kilobase) DNA strands, a capture rate of one target molecule per 80,000 seconds is projected. Thus, unless the number of molecules in the vicinity of the nanopore is somehow increased, a reasonable sample of unamplified DNA would have a single molecule capture rate or nanopore/molecule interaction of order one per day.
Another approach that effectively increases the number of molecules is to reduce the volume of solution in the measurement apparatus while keeping the number of molecules the same. This can be affected by a conventional technique such as centrifuging the sample prior to inserting it into the sequencing apparatus. However, this introduces an additional step into the work flow that adds cost. Furthermore, when the initial number of target molecules is small, such as when collecting a pathogen from the environment, preconcentration has the significant downside of reducing the sample volume to potentially an unacceptable small level. For example, if the starting sample volume is 10 μl then concentrating it by a factor of 1000 would result in a volume of 0.01 μl. Such a sample requires an exceptionally small analyte chamber, requiring advanced fabrication and fluid handling technology.
Furthermore, to achieve an acceptable sequencing throughput, it is expected that an array of nanopores will be required. The present state of technology suggests a nanopore sequencing rate of order 10,000 bases per second. To sequence the 3 billion bases of the human genome in three hours, it would require at least 30 nanopores and likely much more (500 nanopores) if the DNA is broken into smaller sub strands. An array of such pores would require an analyte volume much larger than for a system that contained a single nanopore. Thus, the conventional step in the prior practice of preconcentrating the sample to a smaller volume makes constructing an array of sequencing elements within the same analyte volume all the more challenging.
Generally, the arrival rate of the target molecules at a detection element or nanopore is limited by the diffusion rate of the molecule in solution. A possible solution would thus be to increase the diffusion rate of the target molecules, for example by increasing the temperature of the electrolyte or increasing the molecular mobility. However, such modifications may not be compatible with the method used to identify the molecule, and in particular may be very deleterious. For a more detailed discussion of the problems associated with diffusion of target molecules, see U.S. patent application Ser. No. 12/395,682 entitled “System and Method to Improve Sequencing Accuracy of a Polymer” filed Mar. 1, 2009.
Accordingly, a method is needed to increase the number of target molecules within the vicinity of a nanopore without pre-concentrating the sample, and thereby reducing its volume, and without increasing the absolute number of analyte molecules by molecular amplification. Such a method must be compatible with the constraints imposed by the sensing system and detection element, for example it should not add to the noise level of the sensing system, and must be compatible with the voltage and pressure tolerances of the detection element.